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PMID:15198992

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Citation

Ilkovski, B, Nowak, KJ, Domazetovska, A, Maxwell, AL, Clement, S, Davies, KE, Laing, NG, North, KN and Cooper, ST (2004) Evidence for a dominant-negative effect in ACTA1 nemaline myopathy caused by abnormal folding, aggregation and altered polymerization of mutant actin isoforms. Hum. Mol. Genet. 13:1727-43

Abstract

We have studied a cohort of nemaline myopathy (NM) patients with mutations in the muscle alpha-skeletal actin gene (ACTA1). Immunoblot analysis of patient muscle demonstrates increased gamma-filamin, myotilin, desmin and alpha-actinin in many NM patients, consistent with accumulation of Z line-derived nemaline bodies. We demonstrate that nebulin can appear abnormal secondary to a primary defect in actin, and show by isoelectric focusing that mutant actin isoforms are present within insoluble actin filaments isolated from muscle from two ACTA1 NM patients. Transfection of C2C12 myoblasts with mutant actin(EGFP) constructs resulted in abnormal cytoplasmic and intranuclear actin aggregates. Intranuclear aggregates were observed with V163L-, V163M- and R183G-actin(EGFP) constructs, and modeling shows these residues to be adjacent to the nuclear export signal of actin. V163L and V163M actin mutants are known to cause intranuclear rod myopathy, however, intranuclear bodies were not reported in patient R183G. Transfection studies in C2C12 myoblasts showed significant alterations in the ability of V136L and R183G actin mutants to polymerize and contribute to insoluble actin filaments. Thus, we provide direct evidence for a dominant-negative effect of mutant actin in NM. In vitro studies suggest that abnormal folding, altered polymerization and aggregation of mutant actin isoforms are common properties of NM ACTA1 mutants. Some of these effects are mutation-specific, and likely result in variations in the severity of muscle weakness seen in individual patients. A combination of these effects contributes to the common pathological hallmarks of NM, namely intranuclear and cytoplasmic rod formation, accumulation of thin filaments and myofibrillar disorganization.

Links

PubMed Online version:10.1093/hmg/ddh185

Keywords

Actins/genetics; Biopolymers/genetics; Blotting, Western; Cells, Cultured; DNA Primers; Histological Techniques; Humans; Immunoblotting; Immunohistochemistry; Isoelectric Focusing; Models, Molecular; Muscle Proteins/metabolism; Mutation/genetics; Myoblasts/metabolism; Myopathies, Nemaline/genetics; Protein Folding; Protein Isoforms/genetics; Transfection

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:ACTS

located_in

GO:0015629: actin cytoskeleton

ECO:0000315: mutant phenotype evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:ACTS

located_in

GO:0001725: stress fiber

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:ACTS

located_in

GO:0005865: striated muscle thin filament

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:ACTS

part_of

GO:0015629: actin cytoskeleton

ECO:0000315: mutant phenotype evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:ACTS

part_of

GO:0001725: stress fiber

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:ACTS

part_of

GO:0005865: striated muscle thin filament

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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